A real‐time PCR toolbox for accurate identification of invasive fruit fly species

Significant plant pests such as fruit flies that travel with fresh produce between countries as eggs or larvae pose a great economic threat to the agriculture and fruit industry worldwide. Time‐limited and expensive quarantine decisions require accurate identification of such pests. Immature stages are often impossible to identify, making them a serious concern for biosecurity agencies. Use of COI barcoding PCR, often the only molecular identification resource, is time‐consuming. We assess the suitability of the COI barcoding region for real‐time PCR assays to identify four pest fruit fly species (Family: Tephritidae), in a diagnostic framework. These species, namely Mediterranean fruit fly (Ceratitis capitata), Queensland fruit fly (Bactrocera tryoni), African invader fly (Bactrocera invadens) and Island fly (Dirioxa pornia) each provide a different set of genetic species delimitation problems. We discuss the benefits and limitations of using a single‐gene TaqMan^? real‐time approach for such species. Our results indicate that COI‐based TaqMan^? real‐time PCR assays, in particular for genetically distinct species, provide an accurate, sensitive and rapid diagnostic tool.     

Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene,AvrLmS‐Lep2

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene‐for‐gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus ‘Surpass 400’. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map‐based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas‐LepR2, and an RlmS‐line. The gene, renamed AvrLmS‐Lep2, encodes a small cysteine‐rich protein of unknown function with an N‐terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS‐Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS‐Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.     

Distribution of some triterpenes and phenolic compounds in the extractives of endemic dipterocarpaceae species of Sri Lanka

Extractives of bark and/or timber of 11 species belonging to the genera Cotylelobium, Hopea, Shorea, Vateria and Vatica yielded a fatty-acid ester, a sitosteryl ester, β-amyrin acetate, β-amyrin, dipterocarpol, ursolic acetate, lupeol, sitosterol, ursolic acid, betulinic acid, hexamethyl-coruleoellagic acid, tetramethylellagic acid, chrysophanol and scopoletin. The distribution of these compounds in 18 other species was examined by TLC screening.     

Leptosphaeria maculans AvrLm9: a new player in the game of hide and seek with AvrLm4‐7

Blackleg disease of Brassica napus caused by Leptosphaeria maculans (Lm) is largely controlled by the deployment of race‐specific resistance (R) genes. However, selection pressure exerted by R genes causes Lm to adapt and give rise to new virulent strains through mutation and deletion of effector genes. Therefore, a knowledge of effector gene function is necessary for the effective management of the disease. Here, we report the cloning of Lm effector AvrLm9 which is recognized by the resistance gene Rlm9 in B. napus cultivar Goéland. AvrLm9 was mapped to scaffold 7 of the Lm genome, co‐segregating with the previously reported AvrLm5 (previously known as AvrLmJ1). Comparison of AvrLm5 alleles amongst the 37 re‐sequenced Lm isolates and transgenic complementation identified a single point mutation correlating with the AvrLm9 phenotype. Therefore, we renamed this gene as AvrLm5‐9 to reflect the dual specificity of this locus. Avrlm5‐9 transgenic isolates were avirulent when inoculated on the B. napus cultivar Goéland. The expression of AvrLm5‐9 during infection was monitored by RNA sequencing. The recognition of AvrLm5‐9 by Rlm9 is masked in the presence of AvrLm4‐7, another Lm effector. AvrLm5‐9 and AvrLm4‐7 do not interact, and AvrLm5‐9 is expressed in the presence of AvrLm4‐7. AvrLm5‐9 is the second Lm effector for which host recognition is masked by AvrLm4‐7. An understanding of this complex interaction will provide new opportunities for the engineering of broad‐spectrum recognition.     

Preliminary phenotypic and molecular screening for potential bacterial biocontrol agents of Leptosphaeria maculans, the blackleg pathogen of canola

Leptosphaeria maculans causes blackleg disease of canola (Brassica napus L.). Bacteria isolated from soil, canola stubble and plant parts were assayed for suppression of blackleg. In plate assays, the bacteria isolated from canola stubble had the highest agar-diffusible antifungal activity (75%), which was fungitoxic. In plant cotyledon assays, endophytes had the highest disease suppression. Bacteria with the highest disease suppression in cotyledon assays also had significant disease suppression at the three- to four-leaf stage. PCR screening for bacterial biosynthetic genes, commonly thought to be involved in plant disease suppression, revealed 22 bacteria to be positive for pyrrolnitrin. Pseudomonas chlororaphis and P. aurantiaca isolates contained the phenazine biosynthetic gene. Three Bacillus cereus isolates had the zmaR resistance gene. This study generated a novel set of primers specific to the zwittermicin A biosynthetic cluster. The PCR screening has confirmed the presence of genes encoding pyrrolnitrin (55%), phenazine (10%), zwittermicin A biosynthesis (7.5%) and zwittermicin A resistance (7.5%) from the canola phyllosphere and rhizosphere, which seems more widely distributed than genes for 2,4-diacetylphloroglucinol and pyoluteorin.     

Beta- Lactam Antibiotics Stimulate Biofilm Formation in Non-Typeable Haemophilus influenzae by Up-Regulating Carbohydrate Metabolism

Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10 µg/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended.     

Ultrastructural localisation of alkaline phosphatase in the intertubular tissue of the testis in the domestic fowl

Alkaline phosphatase activity in the intertubular tissue of the testes of the domestic fowl was examined using an ultracytochemical technique based on the lead capture method. In the interstitial tissue, the Leydig cells, transitional cells and the fibroblasts displayed enzyme activity on their cell membranes. Vacuoles located in the transitional cells were lined by reaction products of enzyme activity, whereas the vacuoles representing extracted lipid droplets and present mainly in the Leydig cells were free of enzyme activity. In the peritubular tissue the cell processes of fibroblasts showed enzyme activity on the cell membranes and in pinocytotic vesicles. Cell processes lying adjacent to blood vessels showed pronounced activity. In the blood vessel itself some activity was present in the basement membrane and the endothelium. The surface of the red blood cell showed moderate activity. The possible role of alkaline phosphatase in the transfer of hormone from the Leydig cells to the seminiferous tubules and from the seminiferous tubules to the interstitium is discussed. The myoid cells and their processes were devoid of enzyme activity.     

Real‐time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine application

Zeugodacus cucumis and Bactrocera jarvisi are pests of fruit and vegetable crops and cause damage to horticulture industries. Immature stages of these two fruit fly species have been intercepted in New Zealand a number of times. Identification to species was not possible using morphological characters; thus, it is important to develop an assay for their species‐level identification. Here, the real‐time PCR assays for rapid identification of Z. cucumis and B. jarvisi were developed and validated. The PCR protocols demonstrated their specificity by amplifying the two target species successfully, with no cross‐reactions observed in the tested tephritid species. The in silico test of the primer and probe binding sites of the two assays also demonstrated the assays’ specificity by no mismatches present in the binding regions of the target species, but 1–4 mismatches in the binding regions of the non‐target fruit fly species. The thresholds of detection for the two assays are as low as 10 copies/µl of the target DNA, indicating that the assays have a very high sensitivity. The application of the real‐time PCR assays has greatly assisted in routine pest identifications at the New Zealand border and surveillance programme. Therefore, the assays have the potential to be used by diagnostic agencies and research organizations worldwide.     

The Human Tau Interactome: Binding to the Ribonucleoproteome,and Impaired Binding of the Proline-to-Leucine Mutant at Position 301 (P301L) to Chaperones and the Proteasome

The tau protein is central to the etiology of several neurodegenerative diseases, including Alzheimer''s disease, a subset of frontotemporal dementias, progressive supranuclear palsy and dementia following traumatic brain injury, yet the proteins it interacts with have not been studied using a systematic discovery approach. Here we employed mild in vivo crosslinking, isobaric labeling, and tandem mass spectrometry to characterize molecular interactions of human tau in a neuroblastoma cell model. The study revealed a robust association of tau with the ribonucleoproteome, including major protein complexes involved in RNA processing and translation, and documented binding of tau to several heat shock proteins, the proteasome and microtubule-associated proteins. Follow-up experiments determined the relative contribution of cellular RNA to the tau interactome and mapped interactions to N- or C-terminal tau domains. We further document that expression of P301L mutant tau disrupts interactions of the C-terminal half of tau with heat shock proteins and the proteasome. The data are consistent with a model whereby a higher propensity of P301L mutant tau to aggregate may reflect a perturbation of its chaperone-assisted stabilization and proteasome-dependent degradation. Finally, using a global proteomics approach, we show that heterologous expression of a tau construct that lacks the C-terminal domain, including the microtubule binding domain, does not cause a discernible shift of the proteome except for a significant direct correlation of steady-state levels of tau and cystatin B.The tau protein is a member of the family of microtubule-associated proteins (MAPs)¹ that in humans is coded by the MAPT gene on chromosome 17q21.31 (1). Initially, described as a factor that binds to and stabilizes microtubules (MTs) (2), interest in the tau protein grew when it was shown to represent the main constituent of intracellular protein aggregates, termed neurofibrillary tangles (NFTs), observed in Alzheimer''s disease (3, 4). Similar tau aggregates have since been described in other, less common dementias, including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick''s disease and dementia pugilistica, a form of dementia observed in athletes who had been exposed to repeated traumatic brain injury (5).Despite its early recognition as a MT binding molecule, the physiological function of the tau protein is still being debated (6). At least, in part, this uncertainty is born from the observation that tau knockout mice are rather nonconspicuous in their phenotype (7, 8). Ongoing attempts to define additional roles for this protein have, over the years, generated several hypotheses, including that the tau protein modulates neurite outgrowth and axonogenesis (8, 9), bridges the microtubule and actin cytoskeletons (10), and acts as a scaffold for tethering the Src family tyrosine kinase Fyn to PSD-95/NMDA receptor complexes (11). The predominant expression of tau in neuronal axons suggests a role in brain function. Significantly, in all tauopathies, a group term used to describe dementias with pathological tau protein involvement, the tau protein is observed to detach from microtubules and to form aggregates. There also is compelling evidence from a body of work with transgenic models that the cellular toxicity observed in the aforementioned dementias relies on the presence of the tau protein (12). Consequently, it seems plausible that the cellular toxicity observed in AD and other dementias does not relate to a loss of function of the tau protein but represents a gain of toxic function the protein exhibits in its microtubule-detached form.The tau molecule can be crudely subdivided into an amino-terminal projection domain (PD), a microtubule-binding domain (MTB), and a carboxy-terminal domain (C'') (13). The protein has long been known to exhibit some remarkable biochemical characteristics, including an ability to withstand harsh acid and heat treatments that would cause a majority of other proteins to precipitate (2, 14). These characteristics have been attributed to tau being natively unfolded and possessing a highly dynamic character (15). The tau protein is also known to be a substrate for several post-translational modifications (PTMs), and the list of tau modifying enzymes that have been described is long. In particular, tau phosphorylation has been recognized to occur in vivo and in disease, and tau hyperphosphorylation at sites within the MTB domain and at nearby sites flanking the MTB has been shown to promote detachment of tau from microtubules (16). There further is broad agreement in the field that levels of several other tau PTMs are raised in tauopathies, including nitration (17), ubiquitination (18), sumoylation (19), and truncation (20, 21). Less agreement exists on the degree to which specific PTMs contribute to disease manifestation in individual tauopathies (22). Lacking also are insights into other physiological protein interactions the tau protein engages in and, surprisingly, to our knowledge, no systematic screen for tau binders has been reported. Thus, except for its well-established binding to microtubules (2), members of the Src family of protein kinases (23, 24), Hsp70 (25)/Hsp90 (26, 27), and reports on its interaction with F-actin (28), ApoE3 (29), a subset of peptidyl-prolyl cis-trans isomerases (30, 31), α-synuclein (32), PACSIN1 (33), and negatively charged polymers, including nucleic acids (34, 35), relatively little is known about other nonenzymatic interactions the protein engages in.In an attempt to address this unmet need, we set out to define molecular interactors of the tau protein in the human neuroblastoma SH-SY5Y cell model. The study made use of advanced instrumentation and workflows that included comparative mass spectrometry based on isobaric tags. We observed that the tau interactome is dramatically enriched in cellular components involved in the regulation and execution of RNA-processing and translation. We document that the previously known ability of the tau protein to bind to nucleic acids is partially responsible but not sufficient by itself to explain this binding propensity of the tau protein. We narrowed down the binding preference of individual binders to N- and C-terminal domains within tau and document that several interactors, including 14-3-3 proteins, heat shock proteins, and the proteasome, exhibit a strong binding preference for the C terminus of tau. When comparing the interactomes of wild-type and mutant tau (P301L) linked to frontotemporal dementia, we observed that interactions with the aforementioned C-terminal tau binding partners are diminished for mutant tau. Despite the strong binding of tau to the ribonucleoproteome, its overexpression does not seem to affect the global steady-state levels of cellular proteins, and only the levels of cystatin B, a natural inhibitor of cysteine proteases, were modified and correlated directly with the levels of heterologously expressed tau.